My long-term research goal is to study the mechanisms of virulence in the intracellular pathogen Toxoplasma gondii, and determine the role of parasite strain type in determining disease outcome. T. gondii is a member of the phylum Apicomplexa which includes other human parasites, including the causative agents of malaria and cryptosporidiosis. In humans Toxoplasma is an opportunistic pathogen, and infections are particularly severe in the developing fetus and in individuals who have become immunocompromised. While it was known that different strains of Toxoplasma caused different disease in mice (and possibly humans), until recently the parasite proteins responsible had not been identified. We, and others, have recently identified a parasite-derived secreted protein kinase (rhoptry protein 18;ROP18) that increases the lethality of avirulent Toxoplasma in mice by over 4 logs. While the effect of ROP18 on virulence in mice is clear, its mechanism of action is poorly understood, and it is the goal of this project to determine the molecular and cellular mechanisms for the impact of ROP18 on virulence. To do this two complementary approaches are described. In Aim 1 I will identify the cellular and molecular signaling events that are manipulated by ROP18 during intraperitoneal infection in vivo. I will identify transcriptional, cytokine secretion, and immune cell type recruitment differences in mice infected with strains expressing virulent and avirulent forms of ROP18, and correlate them with disease progression using in vivo bioluminescence imaging. In Aim 2 I will develop small-molecule probes for the kinase domain of ROP18 to allow for the direct manipulation of ROP18 kinase activity in vivo and for the identification of the physiological substrates of ROP18. These approaches should yield new insights into the molecular targets of ROP18 that are relevant to its effects on virulence, and increase our understanding of the mechanisms of virulence in general. Moreover they may result in the development of molecular markers that would allow the severity of human Toxoplasma infections to be predicted so that treatment regimens could be tailored accordingly. RELEVANCE (See instructions): Toxoplasmosis is a disease that can be very severe in AIDS patients and the developing fetus, and it appears that the severity of infection is at least partially dependent on Toxoplasma strain type. The goal of this project is to understand why some strains of Toxoplasma cause more severe disease than others. This will improve our ability to diagnose more aggressive infections and prescribe treatments more effectively.